甲基CpG结合域四聚体蛋白的原核表达、纯化及鉴定

目的 在大肠埃希菌中表达甲基CpG结合域四聚体蛋白，并对其进行纯化和鉴定。方法 将重组表达质粒4×MBD-pET30b+转化大肠埃希菌DH5a克隆扩增并测序鉴定后，转化大肠埃希菌BL21（DE3）后接种于LB 培养基中，异丙基硫代半乳糖苷（IPTG）诱导重组蛋白的表达, 表达产物经Ni-NTA亲和层析纯化，通过SDS-PAGE和Western blot鉴定蛋白表达，用此蛋白免疫染色人胚肾细胞后用荧光显微镜观察。结果 克隆质粒测序结果与理论预期完全一致，SDS-PAGE显示在大肠埃希菌中表达出相对分子量为46 000的目标蛋白，Western blot显示目标蛋白带有His融合标签（氨基端）和HA标签（羧基端）。荧光显微镜观察显示MBD蛋白能与细胞内的甲基化CpG基序特异性结合。结论 成功表达并纯化了具有免疫原性的甲基CpG结合域四聚体蛋白，为今后进一步研究DNA甲基化奠定了基础。

Prokaryotic expression, purification and identification of tetrameric-protein of methyl-CpG-binding domain

Objective To express, purify and identify tetrameric protein of methyl-CpG-binding domain in E. coli. Methods The recombinant plasmid 4 × MBD-pET30b + were transformed into E. coli DH5a for clonal expansion and sequenced, then the tetrameric-proteins were expressed in E. coli BL21 (DE_3) under the induction of IPTG. Moreover, the expression products were purified by Ni-NTA chromatography, and were determined by SDS-PAGE and Western blot. Immunostain 293T cells with the proteins were analyzed by fluorescence microscope. Results The sequence analysis showed orientation right and was identical with the expectation. SDS-PAGE and Western blot demonstrated that the molecular weight of the tetrameric- protein was 46 0110 with the N-terminal His-tag and the C-terminal HA-tag. The MBD proteins can bind to the intracellular CpG DNA specifically.Conclusions The tetrameric-proteins of methyl-CpG-binding domain are successfully expressed and purified in E. coli. This results establish a groundwork for the further researches on DNA methylation.